Avicenna J Med Biotech

arij002

Avicenna Journal of Medical Biotechnology

2008-2835 2008-4625

Avicenna Research Institute

ajmb40480

Construction and Evaluation of Short Hairpin RNAs for Knockdown of Metadherin mRNA

Zare

Farahnaz

Sharifzadeh

Sedigheh

Behzad-Behbahani

Abbas

Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

Rafiei Dehbidi

Gholamreza

Yousefi

Zahra

Ranjbaran

Reza

Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Seyyedi

Noorossadat

13 4 217 222 7 11 2020 4 4 2021

Background: Short hairpin RNA (shRNA) has proven to be a powerful tool to study genes’ function through RNA interference mechanism. Three different methods have been used in previous studies to produce shRNA expression vectors including oligonucleotide-based cloning, polymerase chain reaction (PCR)-based cloning, and primer extension PCR approaches. The aim of this study was designing a reliable and simple method according to the primer extension strategy for constructing four shRNA vectors in order to target different regions of Metadherin (MTDH) mRNA in human leukemic cell line Jurkat. Methods: Oligonucleotides for construction of four shRNA vectors were designed, synthesized and fused to U6 promoter. Each U6-shRNA cassette was cloned into a pGFP-V-RS vector. MTDH shRNAs were transfected into the Jurkat cell line by using the electroporation method. The ability of shRNAs to knock down MTDH mRNA was analyzed through qRT-PCR. Apoptosis assay was used to evaluate the effect of down regulation of MTDH expression on cell integrity. Results: A significant reduction (about 80%) in the expression levels of MTDH mRNA and an increase in the percentages of apoptotic cells (about 20%) were observed in the test group in comparison with control. Conclusion: MTDH shRNA constructs effectively inhibited gene expression. However, simplicity and inexpensiveness of the method were additional advantages for its application.